[Eosinophilic esophagitis in children: analysis of 22 cases].

Objective: Eosinophilic esophagitis (EoE) is a chronic immune-mediated esophageal disease.The current domestic reports of EoE in children is rare.The aim of this study was to analyze the clinical features, the diagnosis and treatment advance of EoE in children by case analysis and literature review. Method: Clinical data of 22 children with EoE from January, 2011 to December, 2015 in Children's Hospital, Zhejiang University School of Medicine were recorded, retrospective analysis was performed on clinical presentation, gastroendoscopy and histopathological examination features and the treatment. Result: (1) Clinical data: EoE can occur at any age in children (5 months to 13 years). The most common clinical manifestations of EoE are vomiting and abdominal pain, 45% (10/22) and 41%(9/22) respectively. (2) Endoscopy and pathological features of esophageal mucosa: 11 cases with coarse mucous membrane (50%), 6 cases with congestion or erosion of esophageal membrane (27%), 5 cases with longitudinal crack (23%), 3 cases with ring uplift (14%), 3 cases with granular uplift (14%), 3 cases with normal mucosa(14%). Histopathologic manifestation is eosinophil infiltration and the eosinophil counts were all more than or equal to 15/HP. (3) Laboratory results: 13 cases had increasing eosinophil counts and eosinophils proportion (62%). (4)Allergy history: among 22 cases, 7 patients had allergy history (32%). (5) Situation of treatment and remission: 16 cases had clinical remission by oral omeprazole; 2 cases had clinical remission by oral Omeprazole and Montelukast sodium; 1 case acquired remission by elimination diet; 1 case acquired remission by elimination diet and oral prednisone. 2 cases dropped out; Only 2 patients received gastroendoscopy re-examination after 3 months and revealed esophageal mucosal histologic complete recovery. Conclusion: The clinical symptoms of EoE in children varies.Esophageal mucosal features of gastroendoscopy examination in children with EoE were longitudinal crack, white exudates or plaques, paper mucosa, ring uplift and granular uplift.Most patients could achieve remission by using proton-pump inhibitors, only few children needed elimination diet and change formula, or even oral glucocorticoids.

Utilization of Western medicine and traditional Chinese medicine among patients with Alzheimer's disease in Taiwan: a nationwide population-based study.

BACKGROUND AND PURPOSE: Large-scale studies of utilization of medical services among patients with Alzheimer's disease (AD) are lacking. We aimed to investigate the usage of Western medicine and traditional Chinese medicine (TCM) among these patients in Taiwan. METHODS: We analyzed one million samples from the National Health Insurance Research Database in Taiwan. Patients (n = 1814) newly diagnosed with AD in 2001-2010 were divided into TCM users (n = 528) and non-TCM users (n = 1286). RESULTS: Compared with non-TCM users, TCM users were younger, had a higher female:male ratio and higher utilization rate of Western medicine. The median interval between diagnosis and the first TCM consultation was 7.92 months. Donepezil and rivastigmine were commonly prescribed medications. Chinese herbal medicine was the most popular treatment among TCM users. CONCLUSIONS: This study revealed the specific usage patterns of TCM and non-TCM medical services among patients with AD. The information could be used for improving the healthcare of patients with AD.

Generation and characterization of aptamers targeting factor XIa.

BACKGROUND: The plasma protease factor XIa (FXIa) has become a target of interest for therapeutics designed to prevent or treat thrombotic disorders. METHODS: We used a solution-based, directed evolution approach called systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers that target the FXIa catalytic domain. RESULTS: Two aptamers, designated 11.16 and 12.7, were identified that bound to previously identified anion binding and serpin bindings sites on the FXIa catalytic domain. The aptamers were non-competitive inhibitors of FXIa cleavage of a tripeptide chromogenic substrate and of FXIa activation of factor IX. In normal human plasma, aptamer 12.7 significantly prolonged the aPTT clotting time. CONCLUSIONS: The results show that novel inhibitors of FXIa can be prepared using SELEX techniques. RNA aptamers can bind to distinct sites on the FXIa catalytic domain and noncompetitively inhibit FXIa activity toward its primary macromolecular substrate factor IX with different levels of potency. Such compounds can be developed for use as therapeutic inhibitors.

Evidence for factor IX-independent roles for factor XIa in blood coagulation.

BACKGROUND: Factor XIa is traditionally assigned a role in FIX activation during coagulation. However, recent evidence suggests this protease may have additional plasma substrates. OBJECTIVE: To determine whether FXIa promotes thrombin generation and coagulation in plasma in the absence of FIX, and to determine whether FXI-deficiency produces an antithrombotic effect in mice independently of FIX. METHODS: FXIa, FXIa variants and anti-FXIa antibodies were tested for their effects on plasma coagulation and thrombin generation in the absence of FIX, and for their effects on the activation of purified coagulation factors. Mice with combined FIX and FXI deficiency were compared with mice lacking either FIX or FXI in an arterial thrombosis model. RESULTS: In FIX-deficient plasma, FXIa induced thrombin generation, and anti-FXIa antibodies prolonged clotting times. This process involved FXIa-mediated conversion of FX and FV to their active forms. Activation of FV by FXIa required the A3 domain on the FXIa heavy chain, whereas activation of FX did not. FX activation by FXIa, unlike FIX activation, was not a calcium-dependent process. Mice lacking both FIX and FXI were more resistant to ferric chloride-induced carotid artery occlusion than FXI-deficient or FIX-deficient mice. CONCLUSION: In addition to its predominant role as an activator of FIX, FXIa may contribute to coagulation by activating FX and FV. As the latter reactions do not require calcium, they may make important contributions to in vitro clotting triggered by contact activation. The reactions may be relevant to FXIa's roles in hemostasis and in promoting thrombosis.

Partial protective of chickens against Eimeria tenella challenge with recombinant EtMIC-1 antigen.

Eimeria tenella microneme protein 1 (EtMIC-1) is highly conserved with TgMIC-2, which is involved in parasite binding specifically to host cells. Little is known about the immune responses and protective efficacy against E. tenella infection with EtMIC-1 antigen. In the present study, the recombinant proteins of E. tenella mature MIC-1 and adhesive domain (von Willebrand factor type A domain, EtMIC-1-VD) were obtained, protective efficacy against E.tenella infection and the mucosal immune response, which is induced in broilers was evaluated. The antibody levels and the transcription profiles of cytokine of chickens, such as interleukin-12 (IL-12) and interferon-γ (IFN-γ), were detected after being immunized three times with the recombinant EtMIC-1 and EtMIC-1-VD by ELISA assay and quantitative real-time PCR, respectively. The results showed that both groups of chickens, after being immunized with 100 µg EtMIC-1 or EtMIC-1-VD antigen, induced about tenfold higher IgG levels compared to the nonimmune groups. The transcription profiles of IL-12 and IFN-γ of the immunized groups were significantly higher than the control groups as well. The anticoccidial index of the group immunized with 100 µg EtMIC-1 and the group immunized with 100 µg EtMIC-1-VD were 167.2 and 165.5, respectively, which are significantly higher than low-dose immunized groups and challenged control groups. Our data suggests that VD domain is the key functional structure of EtMIC-1 that could trigger a significant humoral and cellular response against E. tenella infection, and EtMIC-1 had the potential in imparting partial protection in chickens against homologous challenge.

Analysis of the factor XI variant Arg184Gly suggests a structural basis for factor IX binding to factor XIa.

BACKGROUND: A patient with factor XI (FXI) deficiency was reported with an Arg184Gly substitution in the FXI A3 domain. The A3 domain contains an exosite required for binding of FIX to activated FXI (FXIa). OBJECTIVE: To test the effects of the Arg184Gly substitution on FIX activation, and to characterize the FIX-binding site on FXIa. METHODS: Recombinant FXIa and FIX variants were used to identify residues involved in FIX activation by FXIa. Analysis of the FXI structure was used to identify potential FIX-binding sites. RESULTS: The Km for FIX activation by FXIa-Gly184 was approximately three-fold higher than for FXIa, suggesting that Arg184 is part of the exosite. Arg184 and the adjacent residues, Ile183 and Asp185, contribute to charged and hydrophobic areas that are not present in the FXI homolog prekallikrein (PK). Replacing residues 183-185 with alanine abolished exosite activity, similarly to replacement of the entire A3 domain with the A3 domain from PK (FXIa/PKA3). Reintroducing FXI residues 183-185 into FXIa/PKA3 partially restored the exosite, and replacing residues 183-185 and 260-264 completely restored exosite function. FIX in which the Ω-loop (residues 4-11) was replaced with the FVII Ω-loop was activated poorly by FXIa, suggesting that the FIX Ω-loop binds to FXIa. CONCLUSIONS: The results support a model in which the Ω-loop of FIX binds to an area on FXIa composed of residues from the N-terminus and C-terminus of the A3 domain. These residues are buried in zymogen FXI, and must be exposed upon conversion to FXIa to permit FIX binding.

The occurrence of the feline "Candidatus Mycoplasma haemominutum" in dog in China confirmed by sequence-based analysis of ribosomal DNA.

In the present study, polymerase chain reaction (PCR) assay was used to detect haemoplasmas (haemotropic bacteria) in 40 clinically healthy pet dogs in Foshan city, Guangdong Province, China, and one dog was found positive. Comparison of its 16S ribosomal DNA (rDNA) sequence with relevant sequences showed that the isolated haemoplasma had greater sequence identity to feline species "Candidatus Mycoplasma haemominutum" (99%) than to "Candidatus Mycoplasma haematoparvum" (95%). This result, for the first time, indicates the presence of the feline "Candidatus Mycoplasma haemominutum" in Chinese dogs and it represents the first survey of its kind in China by using PCR assay. The results indicated that dog may represent one of the hosts for the feline "Candidatus Mycoplasma haemominutum".

A cross-reactive material positive variant of coagulation factor XI (FXIP520L) with a catalytic defect.

Inherited deficiency of the trypsin-like protease factor (F) XI is associated with a mild to moderate bleeding diathesis. In most cases, FXI protein is reduced in plasma, and examples of dysfunctional circulating FXI variants are rare. We characterized the defect in one such variant with a proline to leucine substitution at residue 520. FXI Pro520 corresponds to chymotrypsin Pro161, and is conserved in most members of the chymotrypsin protease family. Recombinant FXI containing this substitution will be referred to as FXI(P161L). k(cat) for cleavage of chromogenic substrates and for activation of the natural FXIa substrate FIX is approximately 3-fold lower for activated FXI(P161L) (FXIa(P161L)) than for wild-type FXIa (FXIa(WT)), consistent with an abnormal protease active site. Inhibition of FXIa(P161L) by diisopropyl fluorophosphate is 2.4-fold slower than for FXIa(WT), suggesting distortion of the protease oxyanion hole. Binding to p-aminobenzamidine, a probe for the integrity of the S1 substrate-binding site, was similar for FXIa(WT) and FXIa(P161L). Rates of carbamylation of Ile16 were also similar for FXIa(WT) and FXIa(P161L), indicating that the critical salt bridge between Ile16 and Asp194 forms normally during protease activation. Cumulatively, the data demonstrate that Pro161 is required for normal active site oxyanion hole conformation in FXIa. Examination of the FXIa crystal structure and modeling studies indicate that Pro161 forms several hydrophobic contacts with adjacent amino acids that stabilize active site conformation. Leucine can be incorporated at position 161 in FXIa, but would not form the extensive stabilizing network of hydrophobic interactions formed by Pro161.

Factor XI apple domains and protein dimerization.

The coagulation protease zymogen factor (F)XI is a disulfide bond-linked homodimer, a configuration that is necessary for protein secretion and function. The non-catalytic portion of the FXI polypeptide contains four repeats called apple domains (A1-A4). It is clear that FXI A4 plays a key role in dimer formation, however, the importance of other apple domains to this process has not been examined. We prepared recombinant FXI molecules in which apple domains were exchanged with those of the structurally homologous monomeric protein prekallikrein (PK). As expected, FXI/PK chimeras containing FXI A4 are dimers, while those with PK A4 are monomers. FXI A4 contains cysteine at position 321 that forms the interchain disulfide bond, while Cys321 in PK is unavailable for interchain bond formation because it is paired with Cys326. FXI/PK chimeras containing PK A4 were modified by changing Cys326 to glycine, leaving Cys321 unpaired (PKA4-Gly326). FXI with a PK A4 domain is a monomer, however, introducing PKA4-Gly326 results in a disulfide bond-linked dimer. This indicates that dimer formation can occur in the absence of FXI A4. In proteins containing PKA4-Gly326, replacing FXI A3 with PK A3 partially interferes with dimer formation, while substitution of A2, or A2 and A3 prevents dimer formation. PKA4-Gly326 cannot induce the native PK molecule to dimerize. The data indicate that FXI A2 and A3 make contributions to dimer formation. As these domains are involved in activities that require dimeric protein, it seems reasonable that they stabilize this conformation.

Defective binding of factor XI-N248 to activated human platelets.

Variants of factor XI containing Gln226 to Arg (Q226 to R) and Ser248 to Asn (S248 to N) substitutions were first identified in an African American family with a history of excessive bleeding. The substitutions have recently been identified in unrelated individuals, suggesting they are relatively common. Both amino acids are located in the third apple domain of factor XI, an area implicated in binding interactions with factor IX and activated platelets. Recombinant factor XI-R226 and factor XI-N248 were compared with wild-type factor XI in assays for factor IX activation or platelet binding. Factor XI-R226 activates factor IX with a Michaelis-Menten constant (K(m)) about 5-fold greater than wild-type protein. The catalytic efficiency of factor IX activation is similar to wild-type protein, however, due to an increase in the turnover number (k(cat)) for the reaction. Iodinated factor XI-N248 binds to activated platelets with a dissociation constant (K(d)) more than 5-fold higher than wild-type protein (55 nM and 10 nM, respectively). Activation of factor XI-N248 by thrombin in the presence of activated platelets is slower and does not progress to the same extent as activation of the wild-type protein under similar conditions. Factor XI-N248 activates factor IX normally in a purified protein system and has relatively normal activity in activated partial thromboplastin time (aPTT) assays. Factor XI-N248 is the first factor XI variant described with a clear functional difference compared with wild-type protein. Importantly, the defect in platelet binding would not be detected by routine clinical evaluation with an aPTT assay.

Model for a factor IX activation complex on blood platelets: dimeric conformation of factor XIa is essential.

Human coagulation factor XI (FXI) is a plasma serine protease composed of 2 identical 80-kd polypeptides connected by a disulfide bond. This dimeric structure is unique among blood coagulation enzymes. The hypothesis was tested that dimeric conformation is required for normal FXI function by generating a monomeric version of FXI (FXI/PKA4) and comparing it to wild-type FXI in assays requiring factor IX activation by activated FXI (FXIa). FXI/PKA4 was made by replacing the FXI A4 domain with the A4 domain from prekallikrein (PK). A dimeric version of FXI/PKA4 (FXI/PKA4-Gly326) was prepared as a control. Activated FXI/PKA4 and FXI/PKA4-Gly326 activate factor IX with kinetic parameters similar to those of FXIa. In kaolin-triggered plasma clotting assays containing purified phospholipid, FXI/PKA4 and FXI/PKA4-Gly326 have coagulant activity similar to FXI. The surface of activated platelets is likely to be a physiologic site for reactions involving FXI/FXIa. In competition binding assays FXI/PKA4, FXI/PKA4-Gly326, and FXI have similar affinities for activated platelets (K(i) = 12-16 nM). In clotting assays in which phospholipid is replaced by activated platelets, the dimeric proteins FXI and FXI/PKA4-Gly326 promote coagulation similarly; however, monomeric FXI/PKA4 has greatly reduced activity. Western immunoblot analysis confirmed that activated monomeric FXI/PKA4 activates factor IX poorly in the presence of activated platelets. These findings demonstrate the importance of the dimeric state to FXI activity and suggest a novel model for factor IX activation in which FXIa binds to activated platelets by one chain of the dimer, while binding to factor IX through the other.

The role of high molecular weight kininogen and prothrombin as cofactors in the binding of factor XI A3 domain to the platelet surface.

We have reported that prothrombin (1 microm) is able to replace high molecular weight kininogen (45 nm) as a cofactor for the specific binding of factor XI to the platelet (Baglia, F. A., and Walsh, P. N. (1998) Biochemistry 37, 2271-2281). We have also determined that prothrombin fragment 2 binds to the Apple 1 domain of factor XI at or near the site where high molecular weight kininogen binds. A region of 31 amino acids derived from high molecular weight kininogen (HK31-mer) can also bind to factor XI (Tait, J. F., and Fujikawa, K. (1987) J. Biol. Chem. 262, 11651-11656). We therefore investigated the role of prothrombin fragment 2 and HK31-mer as cofactors in the binding of factor XI to activated platelets. Our experiments demonstrated that prothrombin fragment 2 (1 microm) or the HK31-mer (8 microm) are able to replace high molecular weight kininogen (45 nm) or prothrombin (1 microm) as cofactors for the binding of factor XI to the platelet. To localize the platelet binding site on factor XI, we used mutant full-length recombinant factor XI molecules in which the platelet binding site in the Apple 3 domain was altered by alanine scanning mutagenesis. The recombinant factor XI with alanine substitutions at positions Ser(248), Arg(250), Lys(255), Leu(257), Phe(260), or Gln(263) were defective in their ability to bind to activated platelets. Thus, the interaction of factor XI with platelets is mediated by the amino acid residues Ser(248), Arg(250), Lys(255), Leu(257), Phe(260), and Gln(263) within the Apple 3 domain.

Identification of amino acids in the factor XI apple 3 domain required for activation of factor IX.

Activated coagulation factor XI (factor XIa) proteolytically cleaves its substrate, factor IX, in an interaction requiring the factor XI A3 domain (Sun, Y., and Gailani, D. (1996) J. Biol. Chem. 271, 29023-29028). To identify key amino acids involved in factor IX activation, recombinant factor XIa proteins containing alanine substitutions for wild-type sequence were expressed in 293 fibroblasts and tested in a plasma clotting assay. Substitutions for Ile(183)-Val(191) and Ser(195)-Ile(197) at the N terminus and for Ser(258)-Ser(264) at the C terminus of the A3 domain markedly decreased factor XI coagulant activity. The plasma protease prekallikrein is structurally homologous to factor XI, but activated factor IX poorly. A chimeric factor XIa molecule with the A3 domain replaced with A3 from prekallikrein (FXI/PKA3) activated factor IX with a K(m) 35-fold greater than that of wild-type factor XI. FXI/PKA3 was used as a template for a series of proteins in which prekallikrein A3 sequence was replaced with factor XI sequence to restore factor IX activation. Clotting and kinetics studies using these chimeras confirmed the results obtained with alanine mutants. Amino acids between Ile(183) and Val(191) are necessary for proper factor IX activation, but additional sequence between Ser(195) and Ile(197) or between Phe(260) and Ser(265) is required for complete restoration of activation.

Characterization of a heparin binding site on the heavy chain of factor XI.

The glycosaminoglycan heparin enhances several reactions involving coagulation factor XI (FXI) including activation of FXI by factor XIIa, thrombin, and autoactivation; and inactivation of activated FXI (FXIa) by serine protease inhibitors. We examined the effect of heparin on inhibition of FXIa by the inhibitors C1-inhibitor (C1-INH) and antithrombin III (ATIII). Second order rate constants for inhibition in the absence of heparin were 1.57 x 10(3) and 0.91 x 10(3) M-1 s-1 for C1-INH and ATIII, respectively. Therapeutic heparin concentrations (0.1-1.0 units/ml) enhanced inhibition by ATIII 20-55-fold compared with 0.1-7.0-fold for C1-INH. For both inhibitors, the effect of heparin over a wide range of concentrations (10(-1) to 10(5) units/ml) produced bell-shaped curves, demonstrating that inhibition occurs by a template mechanism requiring both inhibitor and protease to bind to heparin. This implies that FXI/XIa contains structural elements that interact with heparin. Human FXI contains a sequence of amino acids (R250-I-K-K-S-K) in the apple 3 domain of the heavy chain that binds heparin (Ho, D., Badellino, K., Baglia, F., and Walsh, P. (1998) J. Biol. Chem. 273, 16382-16390). To determine the importance of this sequence to heparin-mediated reactions, recombinant FXI molecules with alanine substitutions for basic amino acids were expressed in 293 fibroblasts, and tested in heparin-dependent assays. Inhibition of FXIa by ATIII in the presence of heparin was decreased 4-fold by alanine substitution at Lys253 (A253), with smaller effects noted for mutants A255 and A252. FXI undergoes autoactivation to FXIa in the presence of heparin. The rate of autoactivation was decreased substantially for A253 with modest decreases for A255 and A252. Substituting all four charged residues in the sequence resulted in a profound decrease in autoactivation, significantly greater than for any single substitution. Relative affinity for heparin was tested by determining the concentration of NaCl required to elute FXIa from heparin-Sepharose. Wild type FXIa eluted from the column at 320 mM NaCl, whereas FXIa with multiple substitutions (A252-254 or A250-255) eluted at 230 mM NaCl. All proteins with single substitutions in charged amino acids eluted at intermediate NaCl concentrations. The data indicate that FXI/XIa must bind to heparin for optimal inhibition by ATIII and for autoactivation. Lys253 is the most important amino acid involved in binding, and Lys255 and Lys252 also have roles in interactions with heparin.

Identification of mutations and polymorphisms in the factor XI genes of an African American family by dideoxyfingerprinting.

Congenital deficiency of factor XI is a rare condition associated with a mild to moderate bleeding diathesis that is most commonly found in persons of Jewish ancestry. The disorder has been reported sporadically in a number of other ethnic groups, but rarely in the black population. We report on the genetic analysis of the factor XI genes of two African American patients: a 9-year-old boy (the propositus) with mild factor XI deficiency and his mother. Both individuals have lifelong histories of excessive bleeding. Dideoxyfingerprinting, a technique combining components of single-strand conformational polymorphism analysis and dideoxy-chain termination sequencing, was used in the analysis. Both patients were found to be heterozygous for a mutation changing serine 248 to asparagine [corrected], whereas the propositus was heterozygous for an additional mutation on the paternal allele changing glutamine 226 to arginine. Both mutations reside in the third apple domain of the factor XI heavy chain, an area that has been shown to contain binding sites for factor IX, platelets, and glycosaminoglycans. Previously reported mutations in the factor XI gene seem to cause deficiency primarily by reducing protein expression. Because both alleles in the propositus contain amino acid substitutions, the significant amount of circulating factor XI in his plasma must be comprised entirely of abnormal molecules. Factor XI circulates as a homodimer, and the presence of mutations in both alleles of the factor XI gene suggests that his bleeding disorder is caused in part by the effect of the two abnormal gene products forming dimers in different combinations. Three neutral (not associated with amino acid changes) DNA polymorphisms were also identified in the two subjects: a C to T change at nucleotide 472 in exon 5, A to G at nucleotide 844 in exon 8, and T to C at nucleotide 1234 in exon 11. Analysis of a random sample of normal volunteers showed that these polymorphisms are relatively common, with allele frequencies of 7.4%, 19%, and 18%, respectively. This suggests that there is considerable genetic heterogeneity in the factor XI gene.

A comparison of murine and human factor XI.

Factor XI is a plasma glycoprotein that is required for contact activation initiated fibrin formation in vitro and for normal hemostasis in vivo. In preparation for developing a mouse model of factor XI deficiency to facilitate investigations into this protease's contributions to coagulation, we cloned the complementary DNA for murine factor XI, expressed the protein in a mammalian expression system, and compared its properties with human recombinant factor XI. The 2.8-kb murine cDNA codes for a protein of 624 amino acids with 78% homology to human factor XI. Both recombinant murine and human factor XI are 160 kD homodimers comprised of two 80 kD polypeptides connected by disulfide bonds. Murine factor XI shortens the clotting time of human factor XI deficient plasma in an activated partial thromboplastin time assay, with a specific activity 50% to 70% that of the human protein. In a purified system, murine factor XI is activated by human factor XIIa and thrombin in the presence of dextran sulfate. Murine factor XI differs from human factor XI in that it undergoes autoactivation slowly in the presence of dextran sulfate. This is due primarily to murine factor XIa preferentially cleaving a site on zymogen factor XI within the light chain, rather than the activation site between Arg371 and Val372. Northern blots of polyadenylated messenger RNA show that murine factor XI message is expressed, as expected, primarily in the liver. In contrast, messenger RNA for human factor XI was identified in liver, pancreas, and kidney. The studies show that murine and human factor XI have similar structural and enzymatic properties. However, there may be variations in tissue specific expression and subtle differences in enzyme activity across species.